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SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Protocol Version 2.2.1, February 2012 SureSelect platform manufactured with Agilent SurePrint Technology Research Use Only. Not for use in Diagnostic Procedures. Agilent Technologies Notices © Agilent Technologies, Inc. 2011-2012 Warranty No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws. The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agreement shall control. Manual Part Number G7580-90010 Edition Version 2.2.1, February 2012 Printed in USA Agilent Technologies, Inc. 5301 Stevens Creek Rd Santa Clara, CA 95051 USA Acknowledgement Oligonucleotide sequences © 2006, 2008, and 2011 Illumina, Inc. All rights reserved. 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SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing In this Guide... This guide describes the recommended operational procedures to capture transcripts of interest using the Agilent SureSelect RNA Capture Kit and sample preparation kits for next-generation sequencing. This protocol is specifically developed and optimized to use Biotinylated RNA oligomer libraries, or Bait, to enrich targeted regions of the transcriptome. This guide uses the Illumina paired-end multiplex sequencing platform for library preparation. 1 Before You Begin This chapter contains information (such as procedural notes, safety information, required reagents and equipment) that you should read and understand before you start an experiment. 2 Sample Preparation This chapter describes the steps to prepare the RNA sample for target enrichment. 3 Hybridization This chapter describes the steps to prepare and hybridize samples. 4 Addition of Index Tags by Post-Hybridization Amplification This chapter describes the steps to amplify, purify, and assess quality of the sample library. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 3 What’s New in 2.2 • New product configuration and product numbers for SureSelect reagent kits and capture libraries. • Support for the optional use of the Agilent 2200 TapeStation for RNA quantitation and qualification. • Support for custom SureSelect RNA capture libraries. What’s New in 2.1 • SureSelect RNA Primer Kit for Illumina replaces SureSelect Library Prep Kit. • Dynabeads MyOne Streptavidin T1 beads replaces Agilent LodeStars 2.7 Streptavidin beads. What’s New in 2.0 • Agilent LodeStars 2.7 Streptavidin beads replaces Dynabeads MyOne Streptavidin T1 beads. • NEBNext mRNA Sample Prep Reagent Set 1 replaces the Illumina mRNA-Seq Prep Kit. • QIAquick PCR Purification Kit replaces Agencourt AMPure XP beads for RNA purification. • Agarose gel purification step is added in sample preparation. What’s New in 1.1 • Reagent cap colors are listed where available. • More details given for the reagent kits to use for each step. • Update to cluster generation reagents and procedure. 4 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Content 1 Before You Begin 7 Procedural Notes 8 Safety Notes 8 Required Reagents 9 Optional Reagents 11 Required Equipment 12 Optional Equipment 14 2 Sample Preparation 15 Step 1. Purify the mRNA 18 Step 2. Fragment the RNA 18 Step 3. Clean up the fragmented mRNA 19 Step 4. Synthesize first strand cDNA 20 Step 5. Synthesize second strand cDNA 22 Step 6. Purify the sample with the QIAquick PCR Purification Kit 23 Step 7. Repair the ends 24 Step 8. Purify the sample with the QIAquick PCR Purification Kit 26 Step 9. Add ‘dA’ Bases to the 3' end of the cDNA fragments 27 Step 10. Purify the sample with the QIAquick PCR Purification Kit 28 Step 11. Ligate the paired-end adaptor 29 Step 12. Purify the sample with the QIAquick PCR Purification Kit 30 Step 13. Size-select the DNA fragments with a E-Gel SizeSelect 2% Agarose gel 31 Step 14. Purify the sample with the QIAquick PCR Purification Kit 33 Step 15. Amplify adaptor-ligated library 34 Step 16. Purify the sample with the QIAquick PCR Purification Kit 37 Step 17. Assess quality and quantity with 2100 Bioanalyzer 38 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 5 Contents 3 Hybridization 41 Step 1. Hybridize the library 44 Step 2. Prepare magnetic beads 50 Step 3. Select hybrid capture with SureSelect 51 Step 4. Purify the sample using Agencourt AMPure XP beads 4 Addition of Index Tags by Post-Hybridization Amplification 53 55 Step 1. Amplify the sample to add index tags 56 Step 2. Purify the sample using Agencourt AMPure XP beads 59 Step 3. Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay 60 Step 4. Assess the quantity of each index-tagged library by QPCR 62 Step 5. Pool samples for Multiplexed Sequencing 63 Step 6. Prepare sample for cluster amplification 65 5 Reference 67 SureSelect Reagent Kit Content 68 Other Reagent Kits Content 70 SureSelectXT Indexes for Illumina 73 Alternative Capture Equipment Combinations 6 74 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Protocol 1 Before You Begin Procedural Notes 8 Safety Notes 8 Required Reagents 9 Required Equipment 12 Optional Equipment 14 Make sure you have the most current protocol. Go to the SureSelect Related Literature page on genomics.agilent.com and search for manual number G7580-90010. Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment. NOTE Agilent cannot guarantee the SureSelect Target Enrichment kits and cannot provide technical support for the use of non-Agilent protocols to process samples for enrichment. Agilent Technologies 7 1 Before You Begin Procedural Notes Procedural Notes • Prolonged exposure of SureSelect Elution Buffer to air can decrease product performance by altering the pH of the solution. Keep the Elution Buffer container tightly sealed when not in use. • To prevent contamination of reagents by nucleases, always wear powder-free laboratory gloves and use dedicated solutions and pipettors with nuclease-free aerosol-resistant tips. • Maintain a clean work area. • Do not mix stock solutions and reactions containing RNA on a vortex mixer. Instead, gently tap the tube with your finger to mix the sample. • Avoid repeated freeze-thaw cycles of stock and diluted RNA solutions. • When preparing frozen reagent stock solutions for use: 1 Thaw the aliquot as rapidly as possible without heating above room temperature. 2 Mix briefly on a vortex mixer, then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid. 3 Store on ice or in a cold block until use. • In general, follow Biosafety Level 1 (BL1) safety rules. Safety Notes CAUTION 8 Wear appropriate personal protective equipment (PPE) when working in the laboratory. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Before You Begin Required Reagents 1 Required Reagents Table 1 Required Reagents for Library Prep and Post-Hybridization Amplification Description Vendor and part number DNA 1000 Kit Agilent p/n 5067-1504 High Sensitivity DNA Kit Agilent p/n 5067-4626 Herculase II Fusion DNA Polymerase (includes dNTP mix and 5x Buffer) 200 reactions 400 reactions Agilent Nuclease-free Water (not DEPC-treated) Ambion Cat #AM9930 1X Low TE Buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) Life Technologies p/n 4389764 E-Gel SizeSelect 2% Agarose Gel Life Technologies p/n G6610-02 SuperScript II Reverse Transcriptase Life Technologies 2,000 units 10,000 units 4×10,000 units NEBNext mRNA Sample Prep Reagent Set 1 p/n 600677 p/n 600679 p/n 18064-022 p/n 18064-014 p/n 18064-071 New England BioLabs p/n E6100S Buffer EB (10mM Tris-Cl, ph 8.5) Qiagen p/n 19086 100% Ethanol, molecular biology grade Sigma-Aldrich p/n E7023 3 M NaOAc, pH 5.2 Distilled water SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 9 1 Before You Begin Required Reagents Table 2 Required Reagents for Cluster Generation and Sequencing Description Vendor and part number Illumina Cluster Generation Kit (depending on your instrument and setup) TruSeq PE Cluster Kit v5–CS–GA Illumina p/n PE-203-5001 TruSeq PE Cluster Kit v2–cBot–HS Illumina p/n PE-401-2001 TruSeq PE Cluster Kit v2.5–cBot–HS Illumina p/n PE-401-2510 PhiX Control Kit V2 (for HiSeq 2000) Illumina p/n CT-901-2001 Illumina Sequencing Kit (depending on your instrument and setup) TruSeq SBS Kit v5–GA (36-cycle) Illumina p/n FC-104-5001 TruSeq SBS Kit–HS (50 cycle) Illumina p/n FC-401-1002 Table 3 Reagent Kits 16 Reactions 96 Reactions 480 Reactions SureSelect TE RNA Reagent Kit, HSQ G9601A G9601B G9601C Table 4 10 SureSelect Reagent Kit SureSelect Capture Library (select one) Capture Libraries 16 Reactions 96 Reactions 480 Reactions SureSelect RNA Kinome 5190-4801 5190-4802 5190-4803 SureSelect RNA Capture 1 kb up to 499 Kb 5190-4934 5190-4935 5190-4937 (reorder) 5190-4939 5190-4940 5190-4942 SureSelect RNA Capture 0.5 Mb up to 2.9 Mb 5190-4944 5190-4945 5190-4947 (reorder) 5190-4949 5190-4950 5190-4952 SureSelect RNA Capture 3 Mb up to 5.9 Mb 5190-4954 5190-4955 5190-4957 (reorder) 5190-4959 5190-4960 5190-4962 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Before You Begin Optional Reagents Table 5 1 Required Reagents for Hybridization Description Vendor and part number Dynabeads MyOne Streptavidin T1 Life Technologies 2 mL 10 mL 100 mL Cat #65601 Cat #65602 Cat #65603 Nuclease-free Water (not DEPC-treated) Ambion Cat #AM9930 Optional Reagents Table 6 Optional Reagents Description Vendor and part number Ethylene glycol American Bioanalytical p/n AB00455 RNeasy MinElute Cleanup Kit Qiagen p/n 74204 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 11 1 Before You Begin Required Equipment Required Equipment Table 7 Required Equipment for Library Prep and Post-Hybridization Amplification Description Vendor and part number 2100 Bioanalyzer Agilent p/n G2938C Nuclease-free 1.5 mL microfuge tubes (sustainable at 95°C) Ambion p/n AM12400 or equivalent Thermal cycler Agilent SureCycler, Life Technologies Veriti Thermal Cycler, BioRad (MJ Research) DNA Engine PTC-200, or equivalent Nuclease-free 0.2 mL PCR tubes, thin-walled Eppendorf p/n 951010006 or equivalent Microcentrifuge Eppendorf Microcentrifuge Model 5417C or equivalent P10, P20, P200 and P1000 pipettes Pipetman P10, P20, P200, P1000 or equivalent Vacuum concentrator Savant SpeedVac or equivalent Ice bucket Powder-free gloves Sterile, nuclease-free aerosol barrier pipette tips Timer Vortex mixer Heat block at 37°C 12 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Before You Begin Required Equipment Table 8 1 Required Equipment for Hybridization Description Vendor and part number Mx3000P/Mx3005P 96-well tube plates Agilent p/n 410088 or equivalent Mx3000P/Mx3005P optical strip caps Agilent p/n 401425 or equivalent MicroAmp Clear Adhesive Film Life Technologies p/n 4306311 or equivalent BD Clay Adams Nutator Mixer BD Diagnostics p/n 421105 or equivalent Dynal DynaMag-2 magnetic stand Life Technologies p/n 123-21D or equivalent P10, P20, P200 and P1000 pipettes Pipetman P10, P20, P200, P1000 or equivalent Pipet-Light Multichannel Pipette, 12 channels Rainin p/n L12-20 or equivalent Sterile, nuclease-free aerosol barrier pipette tips Thermal cycler Agilent SureCycler, Life Technologies Veriti Thermal Cycler, BioRad (MJ Research) DNA Engine PTC-200, or equivalent Timer Vortex mixer Water bath or heat block set to 65°C SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 13 1 Before You Begin Optional Equipment Optional Equipment Table 9 Description Vendor and part number Tube-strip capping tool Agilent p/n 410099 Table 10 14 Optional Equipment for Hybridization Optional Equipment for Library Prep and Post-Hybridization Amplification Description Vendor and part number 2200 TapeStation System Agilent p/n G2964AA or G2965AA D1K ScreenTape Agilent p/n 5067-5361 D1K Reagents Agilent p/n 5067-5362 High Sensitivity D1K ScreenTape Agilent p/n 5067-5363 High Sensitivity D1K Reagents Agilent p/n 5067-5364 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Protocol 2 Sample Preparation Step 1. Purify the mRNA 18 Step 2. Fragment the RNA 18 Step 3. Clean up the fragmented mRNA 19 Step 4. Synthesize first strand cDNA 20 Step 5. Synthesize second strand cDNA 22 Step 6. Purify the sample with the QIAquick PCR Purification Kit 23 Step 7. Repair the ends 24 Step 8. Purify the sample with the QIAquick PCR Purification Kit 26 Step 9. Add ‘dA’ Bases to the 3' end of the cDNA fragments 27 Step 10. Purify the sample with the QIAquick PCR Purification Kit 28 Step 11. Ligate the paired-end adaptor 29 Step 12. Purify the sample with the QIAquick PCR Purification Kit 30 Step 13. Size-select the DNA fragments with a E-Gel SizeSelect 2% Agarose gel 31 Step 14. Purify the sample with the QIAquick PCR Purification Kit 33 Step 15. Amplify adaptor-ligated library 34 Step 16. Purify the sample with the QIAquick PCR Purification Kit 37 Step 17. Assess quality and quantity with 2100 Bioanalyzer 38 This section contains instructions for prepped library production specific to the Illumina -read sequencing platform. It is intended for use with the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). Refer to the NEBNext mRNA Sample Prep Reagent Set 1 Instruction Manual for more information. Agilent Technologies 15 2 Sample Preparation Total RNA or mRNA Purify the mRNA mRNA RT 1st strand cDNA 2nd strand synthesis Double-stranded cDNA End repair Blunt-ended fragments with 5'phosphorylated ends Add Klenow and dATP 3'-dA overhang Transcripts Ligate adaptors Bait Design in eArray Adaptor-modified ends SureSelect Oligo Capture Library Removal of unligated adaptors PCR Prepped Library Library Hybridization 24 hours at 65°C Hybrid Capture Selection Magnetic bead selection Amplification PCR and purification Quality Assessment Bioanalyzer and Quantitation Sequencing Figure 1 16 Overall sequencing sample preparation workflow. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Table 11 2 Overview and time requirements Step Time Illumina Prepped library Production 2 days Library Hybridization 25 hours (optional 72 hours) Bead preparation 30 minutes Capture Selection and Washing 2 hours DNA purification 30 minutes Post-Hybridization Amplification 1 hour PCR purification 30 minutes Bioanalyzer QC 1 hour SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 17 2 Sample Preparation Step 1. Purify the mRNA Step 1. Purify the mRNA • Purify 1 to 10 µg of total RNA. Use the purification kit of your choice. Step 2. Fragment the RNA Use reagents from the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S): • NEBNext RNA Fragmentation Buffer (10X) • NEBNext RNA Fragmentation Stop Solution (10X) 1 Preheat a PCR thermal cycler to 94°C. 2 Add the components in Table 12 to a 200 µL thin wall PCR tube. Table 12 Fragmentation Buffer Mix Component Volume NEBNext RNA Fragmentation Buffer (10X)* 2 µL Purified mRNA 1 to 18 µL Nuclease-free Water enough to bring total volume to 20 µL Total 20 µL * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). 3 Incubate the tube in a preheated PCR thermal cycler at 94°C for exactly 5 minutes. 4 Add 2 µL of NEBNext RNA Fragmentation Stop Solution (10X). 5 Put the tube on ice. 18 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 3. Clean up the fragmented mRNA 2 Step 3. Clean up the fragmented mRNA In this step, you precipitate the fragmented mRNA, using reagents from the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). As an alternative, you can use the RNeasy MinElute Cleanup Kit. Elute the RNA in 13.5 µL of Nuclease-Free Water or elution buffer. 1 Transfer the solution to a 1.5 mL RNase-free non-sticky tube. 2 Add the components in Table 13 to the tube and incubate at -80°C for 30 minutes or overnight as desired. Table 13 Component Volume 3 M NaOAC, pH 5.2 2 µL Linear Acrylamide (10 mg/mL)* 1 to 2 µL 100% ethanol 60 µL * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). Stopping Point You can safely stop the protocol here. Store the samples at -15°C to -25°C. Do not stop the protocol at any other point while the sample is RNA. 3 Spin the tube in a microcentrifuge at 14,000 rpm (20,200 relative centrifugal force) for 25 minutes at 4°C. 4 Carefully pipette off the ethanol without dislodging the RNA pellet. The RNA pellets are small and almost colorless. To avoid dislodging the pellets, remove the ethanol in several steps. Remove 90% at each step and switch to smaller pipette tips for each step. 5 Without disturbing the pellet, wash the pellet with 300 µL of 70% ethanol. 6 Spin the pellet in a centrifuge and carefully pipette out the 70% ethanol. 7 Air dry the pellet for 10 minutes at room temperature. 8 Resuspend the RNA in 13.5 µL of Nuclease-free water. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 19 2 Sample Preparation Step 4. Synthesize first strand cDNA Step 4. Synthesize first strand cDNA Use reagents from the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S) and the SuperScript II Reverse Transcriptase. 1 Add the components in Table 14 to a 200 µL thin wall PCR tube: Table 14 Random Primer Mix Component Volume Random Primers (3 µg/µL)* 1 µL mRNA 13.5 µL Total 14.5 µL * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S) 2 Incubate the sample in a PCR thermal cycler at 65°C. Use a heated lid at 105°C. 3 Spin the sample briefly, and then put the tube on ice. 4 Set the PCR thermal cycler to 25°C. 5 Mix the reagents in Table 15 in the order listed in a separate tube. Prepare 10% extra reagent mix if you are preparing multiple samples. Table 15 Random Primer Mix Component Volume for 1 reaction Volume for 10 reactions (with excess) NEBNext First Strand Synthesis Reaction Buffer (5X)* 4 µL 44 µL Murine RNase Inhibitor* 0.5 µL 5.5 µL Total 4.5 µL 49.5 µL * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). 6 Add 4.5 µL of mixture to the PCR tube and mix well. 20 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 4. Synthesize first strand cDNA 2 7 Heat the sample in the preheated PCR thermal cycler at 25°C for 2 minutes. Do not use a heated lid. 8 Add 1 µL of SuperScript II Reverse Transcriptase to the sample. 9 Run the program listed in Table 16 in a thermal cycler to incubate the sample. Use a heated lid at 105°C. Table 16 PCR program Temperature Time 25°C 10 minutes 42°C 50 minutes 70°C 15 minutes 4°C Hold 10 Put the tube on ice. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 21 2 Sample Preparation Step 5. Synthesize second strand cDNA Step 5. Synthesize second strand cDNA Use reagents from NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). 1 Preheat a PCR thermal cycler to 16°C. 2 Add 48 µL of Nuclease-Free Water to the first strand cDNA synthesis mix. 3 Add the reagents the in Table 17 to the mix: Table 17 Component Volume NEBNext Second Strand Synthesis Reaction Buffer (10X)* 8 µL NEBNext Second Strand Synthesis Enzyme Mix* 4 µL * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). 4 Mix well by gentle pipetting. 5 Incubate the sample at 16°C for 2.5 hours in a thermal cycler. Use a heated lid at 50°C. 22 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 6. Purify the sample with the QIAquick PCR Purification Kit 2 Step 6. Purify the sample with the QIAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit (Qiagen p/n 28104). 1 If you haven’t already done so, add the pH Indicator I to the Buffer PB. 2 Add 400 µL of Buffer PB to 80 µL of sample and mix well by pipetting. 3 Check for the yellow color to make sure Buffer PB pH is correct. For more information on how to check buffer pH, refer to the Qiagen QIAquick Handbook. If needed, use 5 µL of the 3M Sodium Acetate (included in the kit) to adjust the pH to the proper range. 4 Put a QIAquick spin column in a 2 mL collection tube. 5 Transfer the 480 µL sample to the QIAquick spin column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 6 Add 750 µL of Buffer PE (concentrate) to the column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 7 Put the QIAquick spin column back in the collection tube (2 mL) and spin in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 8 Transfer the QIAquick spin column to a new 1.5 mL microcentrifuge tube to elute the cleaned sample. 9 Let sit for 2 minutes to completely evaporate residual ethanol. All traces of ethanol must be removed. 10 Add 50 µL of Buffer EB directly onto the QIAquick filter membrane. 11 Wait 60 seconds, then centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 12 Collect the eluate. Stopping Point If you do not continue to the next step, store the samples at -20°C. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 23 2 Sample Preparation Step 7. Repair the ends Step 7. Repair the ends To process multiple samples, prepare master mixes with overage at each step, without the cDNA sample. Master mixes for preparation of 12 samples (including excess) are shown in each table as an example. Prepare the master mix on ice. Use the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). 1 Preheat one heat block to 20°C and the other heat block to 37°C. 2 For 1 library (prepare on ice): • In a sterile microcentrifuge tube, prepare the reaction mix in Table 18. Mix well by gently pipetting up and down. 3 For multiple libraries (prepare on ice): a Prepare the reaction mix in Table 18. Mix well on a vortex mixer. b Add 50 µL of the reaction mix to each 1.5 mL microcentrifuge tube. c Add 50 µL of each DNA sample to each tube. Mix by pipetting. Change pipette tips between samples. 4 Incubate in a thermal cycler for 30 minutes at 20°C. Do not use a heated lid. 24 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 7. Repair the ends Table 18 2 End Repair Mix* Reagent Volume for 1 Library Volume for 12 Libraries (includes excess) Purified double-stranded cDNA sample 50 µL Nuclease-Free Water 25 µL 342.5 µL Phosphorylation Reaction Buffer (10X) 10 µL 125 µL Deoxynucleotide Solution Mix (10 mM each dNTP) 4 µL 20 µL T4 DNA Polymerase 5 µL 62.5 µL DNA Polymerase I, Large (Klenow) Fragment 1 µL 12.5 µL T4 Polynucleotide Kinase 5 µL 62.5 µL Total Volume 100 µL 625 µL (50 µL/sample) * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 25 2 Sample Preparation Step 8. Purify the sample with the QIAquick PCR Purification Kit Step 8. Purify the sample with the QIAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit (Qiagen p/n 28104). 1 If you haven’t already done so, add the pH Indicator I to the Buffer PB. 2 Add 500 µL of Buffer PB to 100 µL of sample and mix well by pipetting. 3 Check for the yellow color to make sure Buffer PB pH is correct. For more information on how to check buffer pH, refer to the Qiagen QIAquick Handbook. If needed, use 5 µL of the 3M Sodium Acetate (included in the kit) to adjust the pH to the proper range. 4 Put a QIAquick spin column in a 2 mL collection tube. 5 Transfer the 600 µL of sample to the QIAquick spin column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 6 Add 750 µL of Buffer PE (concentrate) to the column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 7 Put the QIAquick spin column back in the collection tube (2 mL) and spin in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 8 Transfer the QIAquick spin column to a new 1.5 mL microcentrifuge tube to elute the cleaned sample. 9 Let sit for 2 minutes to completely evaporate residual ethanol. All traces of ethanol must be removed. 10 Add 32 µL of Buffer EB directly onto the QIAquick filter membrane. 11 Wait 60 seconds, then centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 12 Collect the eluate. 26 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 9. Add ‘dA’ Bases to the 3' end of the cDNA fragments 2 Step 9. Add ‘dA’ Bases to the 3' end of the cDNA fragments Use the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). 1 For 1 library (prepare on ice): • In a PCR tube, strip tube, or plate, prepare the reaction mix in Table 19. Mix well by gently pipetting up and down. 2 For multiple libraries (prepare on ice): a Prepare the reaction mix in Table 19. Mix well on a vortex mixer. b Add 18 µL of the reaction mix to each well or tube. c Add 32 µL of each DNA sample to each well or tube. Mix by pipetting. Change pipette tips between samples. Table 19 Adding “A” Bases* Reagent Volume for 1 Library Volume for 12 Libraries (includes excess) Purified end-repaired cDNA sample 32 µL NEBuffer 2 for Klenow Fragment (3’ → 5’ exo-) (10X) 5 µL 62.5 µL Deoxyadenosine 5’- Triphosphate (dATP) (1.0 mM) 10 µL 125 µL Klenow Fragment (3’ → 5’ exo-) 3 µL 37.5 µL Total Volume 50 µL 225 µL (18 µL/sample) * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). 3 Incubate in a thermal cycler for 30 minutes at 37°C. Do not use a heated lid. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 27 2 Sample Preparation Step 10. Purify the sample with the QIAquick PCR Purification Kit Step 10. Purify the sample with the QIAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit (Qiagen p/n 28104). 1 If you haven’t already done so, add the pH Indicator I to the Buffer PB. 2 Add 250 µL of Buffer PB to 50 µL of sample and mix well by pipetting. 3 Check for the yellow color to make sure Buffer PB pH is correct. For more information on how to check buffer pH, refer to the Qiagen QIAquick Handbook. If needed, use 5 µL of the 3M Sodium Acetate (included in the kit) to adjust the pH to the proper range. 4 Put a QIAquick spin column in a 2 mL collection tube. 5 Transfer the 300 µL of sample to the QIAquick spin column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 6 Add 750 µL of Buffer PE (concentrate) to the column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 7 Put the QIAquick spin column back in the collection tube (2 mL) and spin in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 8 Transfer the QIAquick spin column to a new 1.5 mL microcentrifuge tube to elute the cleaned sample. 9 Let sit for 2 minutes to completely evaporate residual ethanol. All traces of ethanol must be removed. 10 Add 23 µL of Buffer EB directly onto the QIAquick filter membrane. 11 Wait 60 seconds, then centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 12 Collect the eluate. Stopping Point 28 If you do not continue to the next step, store the samples at -20°C. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 11. Ligate the paired-end adaptor 2 Step 11. Ligate the paired-end adaptor Use the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S) and the SureSelect RNA Primer Kit. 1 For 1 library (prepare on ice): • In a PCR tube, strip tube, or plate, prepare the reaction mix in Table 20. 2 For multiple libraries (prepare on ice): a Prepare the reaction mix in Table 20. b Add 27 µL of the reaction mix to each well or tube. c Add 23 µL of each cDNA sample to each well or tube. Mix by pipetting. Change pipette tips between samples. Table 20 Ligation master mix Reagent Volume for 1 Library Volume for 12 Libraries (includes excess) Purified dA-Tailed DNA sample 23 µL Quick Ligation Reaction Buffer (2X)* 25 µL 312.5 µL Quick T4 DNA Ligase 1.0 µL 12.5 µL SureSelect Adaptor Oligo Mix (brown cap)† 1.0 µL 12.5 µL Total Volume 50 µL 337.5 µL (27 µL/sample) * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S). † Included in the SureSelect RNA Primer Kit. 3 Incubate for 15 minutes at 20°C on a thermal cycler. Do not use a heated lid. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 29 2 Sample Preparation Step 12. Purify the sample with the QIAquick PCR Purification Kit Step 12. Purify the sample with the QIAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit (Qiagen p/n 28104). 1 If you haven’t already done so, add the pH Indicator I to the Buffer PB. 2 Add 250 µL of Buffer PB to 50 µL of sample and mix well by pipetting. 3 Check for the yellow color to make sure Buffer PB pH is correct. For more information on how to check buffer pH, refer to the Qiagen QIAquick Handbook. If needed, use 5 µL of the 3M Sodium Acetate (included in the kit) to adjust the pH to the proper range. 4 Put a QIAquick spin column in a 2 mL collection tube. 5 Transfer the 300 µL of sample to the QIAquick spin column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 6 Add 750 µL of Buffer PE (concentrate) to the column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 7 Put the QIAquick spin column back in the collection tube (2 mL) and spin in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 8 Transfer the QIAquick spin column to a new 1.5 mL microcentrifuge tube to elute the cleaned sample. 9 Let sit for 2 minutes to completely evaporate residual ethanol. All traces of ethanol must be removed. 10 Add 10 µL of Buffer EB directly onto the QIAquick filter membrane. 11 Wait 60 seconds, then centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 12 Collect the eluate. Stopping Point 30 If you do not continue to the next step, store the samples at -20°C. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 13. Size-select the DNA fragments with a E-Gel SizeSelect 2% Agarose gel 2 Step 13. Size-select the DNA fragments with a E-Gel SizeSelect 2% Agarose gel Use the E-Gel SizeSelect 2% Agarose Gel. 1 Remove a E-Gel SizeSelect 2% Agarose Gel from its package. Remove the combs from the top sample-loading wells and the middle collection wells. Set the E-Gel on the E-Gel iBase linked with the E-Gel Safe Imager. 2 Load the E-Gel as follows: a Load 20 µL of the ligated, purified DNA into a well in the top row. Do not use the center well or outermost wells (to avoid edge effects). Do not load more than 1 µg of DNA. If the sample volume < 20 µL, add nuclease-free water to the well for a total volume of 20 µL. b Load 2 µL 50-bp ladder at 0.1 µg/µL to the center top well. Add 15 µL of water to fill the well. c Fill empty wells in the top row with 20 µL of nuclease-free water. d Fill each of the collection wells in the middle of the gel with 25 µL of nuclease-free water. Add 20 µL of nuclease-free water to the middle center well. 3 Run the gel: • iBase program: Run E-Gel DC • Approximate run time: 13:45 (13 minutes and 45 seconds) Monitor the E-Gel in real-time with the E-Gel® Safe Imager. 4 If needed during the run, fill the middle collection wells with nuclease-free water. 5 When the 200-bp band from the marker lane is in the center of the collection well, stop the run if the run has not already stopped (see Figure 2). 6 Collect the solution from the sample well. 7 Wash each collection well with 25 µL of nuclease-free water, pipette up and down, then retrieve the wash solution and combine with the respective sample solution collected in step 6 for a total of 50 µL. See Table 21 for expected lengths of the insert and PCR according to the excised cDNA length. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 31 2 Sample Preparation Step 13. Size-select the DNA fragments with a E-Gel SizeSelect 2% Agarose gel Figure 2 Table 21 32 Elution of an approximately 200 bp region. This image shows three samples on the same gel. Expected lengths of the insert and PCR according to the excised cDNA length Excised cDNA length (nt) Insert length (bp) PCR product length (bp) 50 ~0 ~100 100 ~50 ~150 150 ~100 ~200 200 ~150 ~250 250 ~200 ~300 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 14. Purify the sample with the QIAquick PCR Purification Kit 2 Step 14. Purify the sample with the QIAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit (Qiagen p/n 28104). 1 If you haven’t already done so, add the pH Indicator I to the Buffer PB. 2 Add 250 µL of Buffer PB to 50 µL of sample and mix well by pipetting. 3 Check for the yellow color to make sure Buffer PB pH is correct. For more information on how to check buffer pH, refer to the Qiagen QIAquick Handbook. If needed, use 5 µL of the 3M Sodium Acetate (included in the kit) to adjust the pH to the proper range. 4 Put a QIAquick spin column in a 2 mL collection tube. 5 Transfer the 300 µL sample to the QIAquick spin column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 6 Add 750 µL of Buffer PE (concentrate) to the column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 7 Put the QIAquick spin column back in the collection tube (2 mL) and spin in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 8 Transfer the QIAquick spin column to a new 1.5 mL microcentrifuge tube to elute the cleaned sample. 9 Let sit for 2 minutes to completely evaporate residual ethanol. All traces of ethanol must be removed. 10 Add 29 µL of Buffer EB directly onto the QIAquick filter membrane. 11 Wait 60 seconds, then centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 12 Collect the eluate. Stopping Point If you do not continue to the next step, store the samples at -20°C. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 33 2 Sample Preparation Step 15. Amplify adaptor-ligated library Step 15. Amplify adaptor-ligated library This step uses PCR to selectively enrich those cDNA fragments that have adaptor molecules on both ends, and to amplify the amount of cDNA in the library. The PCR is done with two primers that anneal to the ends of the adaptors. Ten to fourteen cycles of PCR are used. CAUTION This protocol was optimized to minimize PCR-based bias in the library preparation. While most library preparations yield enough cDNA (100 ng) for at least a single hybridization, poor quality RNA samples or other factors can affect yield. Use reagents in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S), the SureSelect RNA Primer Kit. 1 For 1 library (prepare on ice): • In a PCR tube, strip tube, or plate, prepare the reaction mix in Table 22. Mix well by gently pipetting up and down. 2 For multiple libraries (prepare on ice): a Prepare the reaction mix in Table 22. Mix well on a vortex mixer. b Add 21 µL of the reaction mix to each well or tube. c Add 29 µL of each cDNA sample to each well or tube. Mix by pipetting. Change pipette tips between samples. 34 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 15. Amplify adaptor-ligated library Table 22 2 PCR Components* Reagent Volume for 1 Library Volume for 12 Libraries Size-selected cDNA 29 µL PhusionR HF Buffer (5X) (manufactured by Finnzymes Oy) 10 µL 125 µL SureSelect Primer (brown cap)† 1 µL 12.5 µL SureSelect ILM Indexing Pre Capture PCR Reverse Primer (clear cap)† 1 µL 12.5 µL Deoxynucleotide Solution Mix (10 mM each dNTP) 1.5 µL 18.75 µL Nuclease-Free Water 7 µL 87.5 µL PhusionR High-Fidelity DNA Polymerase (manufactured by Finnzymes Oy) 0.5 µL 6.25 µL Total Volume 50 µL 262.5 µL (21 µL/reaction) * Included in the NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S), except where indicated. † Included in the SureSelect RNA Primer Kit. 3 Run the program listed in Table 23 in a thermal cycler to amplify the sample. Use a heated lid at 105°C. 4 Amplify using the following PCR program: SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 35 2 Sample Preparation Step 15. Amplify adaptor-ligated library Table 23 PCR program Step Temperature Time Step 1 98°C 30 seconds Step 2 98°C 10 seconds Step 3 65°C 30 seconds Step 4 72°C 30 seconds Step 5 36 Repeat Step 2 through Step 4 for a total of 15 times. Step 6 72°C 5 minutes Step 7 4°C Hold SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 16. Purify the sample with the QIAquick PCR Purification Kit 2 Step 16. Purify the sample with the QIAquick PCR Purification Kit Use the reagents from the QIAquick PCR Purification Kit (Qiagen p/n 28104). 1 If you haven’t already done so, add the pH Indicator I to the Buffer PB. 2 Add 250 µL of Buffer PB to 50 µL of sample and mix well by pipetting. 3 Check for the yellow color to make sure Buffer PB pH is correct. For more information on how to check buffer pH, refer to the Qiagen QIAquick Handbook. If needed, use 5 µL of the 3M Sodium Acetate (included in the kit) to adjust the pH to the proper range. 4 Put a QIAquick spin column in a 2 mL collection tube. 5 Transfer the 300 µL of sample to the QIAquick spin column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 6 Add 750 µL of Buffer PE (concentrate) to the column. Spin the sample in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). Discard the flow-through. 7 Put the QIAquick spin column back in the collection tube (2 mL) and spin in a centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 8 Transfer the QIAquick spin column to a new 1.5 mL microcentrifuge tube to elute the cleaned sample. 9 Let sit for 2 minutes to completely evaporate residual ethanol. All traces of ethanol must be removed. 10 Add 30 µL of Buffer EB directly onto the QIAquick filter membrane. 11 Wait 60 seconds, then centrifuge for 60 seconds at 17,900 × g (13,000 rpm). 12 Collect the eluate. Stopping Point If you do not continue to the next step, store the samples at -20°C. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 37 2 Sample Preparation Step 17. Assess quality and quantity with 2100 Bioanalyzer Step 17. Assess quality and quantity with 2100 Bioanalyzer NOTE As an alternative, you can use the D1K ScreenTape (Agilent p/n 5067-5361) and D1K Reagents (Agilent p/n 5067-5362). For more information to do this step, see the Agilent 2200 TapeStation User Manual. Use the Bioanalyzer DNA 1000 to assess the quantity, quality and size distribution of the PCR products. 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide. 2 Open the Agilent 2100 Expert Software (version B.02.02 or higher), turn on the 2100 Bioanalyzer and check communication. 3 Prepare the chip, samples and ladder as instructed in the reagent kit guide. 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation. 5 Within the instrument context, choose the appropriate assay from the drop down list. 6 Start the run. Enter sample names and comments in the Data and Assay context. 7 Verify the results. Check that the electropherogram shows a distribution with a peak size approximately 200 to 250 bp. Measure the concentration of the library by integrating under the peak. 38 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Sample Preparation Step 17. Assess quality and quantity with 2100 Bioanalyzer Figure 3 2 Analysis of amplified prepped library DNA using a DNA 1000 assay. The electropherogram shows a single peak in the size range of 200 to 250 bp. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 39 2 40 Sample Preparation Step 17. Assess quality and quantity with 2100 Bioanalyzer SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Protocol 3 Hybridization Step 1. Hybridize the library 44 Step 2. Prepare magnetic beads 50 Step 3. Select hybrid capture with SureSelect 51 Step 4. Purify the sample using Agencourt AMPure XP beads 53 This chapter describes the steps to combine the prepped library with the hybridization reagents, blocking agents and the SureSelect capture library. CAUTION The ratio of SureSelect capture library to prepped library is critical for successful capture. Agilent Technologies 41 3 Hybridization TOTAL RNA OR mRNA SureSelect™ Target Enrichment System Capture Process NGS Kit + cDNA LIBRARY (PREPPED) + SureSelect HYB BUFFER SureSelect BIOTINYLATED RNA LIBRARY “BAITS” Hybridization STREPTAVIDIN COATED MAGNETIC BEADS + Bead capture UNBOUND FRACTION DISCARDED Wash Beads and Digest RNA Amplify Figure 4 42 Sequencing SureSelect RNA Capture Process SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Hybridization 3 Refer to “SureSelect Reagent Kit Content” on page 68 for a complete content listing of each SureSelect RNA Capture kit. CAUTION You must avoid evaporation from the small volumes of the capture during the 24 hour or greater incubation. If you want to use a different combination of thermal cycler, lid temperature, plates or strips, and sealing method (strip caps or sealing tape), first test the conditions. Incubate 27 µL of SureSelect Hybridization Buffer (without DNA) at 65°C for 24 hours (or longer, if applicable) as a test. Include buffer in each well that you might use, including those in the center and those on the edges. Check that you do not get extensive evaporation. Evaporation should not exceed 3 to 4 µL. For a partial list of tested options showing minimal evaporation, refer to “Alternative Capture Equipment Combinations” on page 74. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 43 3 Hybridization Step 1. Hybridize the library Step 1. Hybridize the library The hybridization reaction requires 100 ng of cDNA with a maximum volume of 3.4 µL. 1 If the prepped library concentration is below 30 ng/µL, use a vacuum concentrator to concentrate the sample at ≤ 45°C. a Add the entire volume of prepped library to an Eppendorf tube. Poke one or more holes in the lid with a narrow gauge needle. You can also break off the cap, cover with parafilm, and poke a hole in the parafilm. b Completely lyophilize. Use a vacuum concentrator on low heat (less than 45°C) to dehydrate. c Reconstitute with nuclease-free water to bring the final concentration to 30 ng/µL (or greater if sample recovery is of concern). Pipette up and down along the sides of the tube for optimal recovery. d Mix well on a vortex mixer and spin in a microfuge for 1 minute. 2 Optional. To test recovery after lyophilization, reconstitute the sample to greater than 30 ng/µL and check the concentration on a Bioanalyzer DNA 1000 chip. See “Step 17. Assess quality and quantity with 2100 Bioanalyzer” on page 38. After quantitation, adjust the sample to 30 ng/µL. Alternatively, concentrate a 100 ng aliquot at ≤ 45°C down to 3.4 µL. If the sample dries up completely, resuspend in 3.4 µL of water and mix on a vortex mixer. If processing multiple samples, adjust to equivalent volumes before concentrating. 3 Mix the components in Table 24 at room temperature to prepare the hybridization buffer. 44 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Hybridization Step 1. Hybridize the library Table 24 3 Hybridization Buffer Reagent Volume for 1 capture (µL), includes excess Volume for 6 captures (µL), includes excess Volume for 12 captures (µL), includes excess SureSelect Hyb #1 (orange cap, or bottle) 25 125 250 SureSelect Hyb #2 (red cap) 1 5 10 SureSelect Hyb #3 (yellow cap) 10 50 100 SureSelect Hyb #4 (black cap, or bottle) 13 65 130 Total 49 (40 µL needed) 245 (40 µL/sample) 490 (40 µL/sample) 4 If precipitate forms, warm the hybridization buffer at 65°C for 5 minutes. 5 In a PCR plate, strip tubes, or tubes, prepare the SureSelect capture library mix for target enrichment: a Keep tubes on ice until step 10. b For each sample, add 5 µL of SureSelect capture library. c For 1 library, combine 1 µL SureSelect RNase Block (purple cap) with 2 µL nuclease-free water. For multiple libraries, use 1 part SureSelect RNase Block (purple cap) to 2 parts nuclease-free water to make enough mix for 2 µL per capture library, plus excess. d Add 2 µL of diluted SureSelect RNase Block (purple cap) to each capture library, and mix by pipetting. 6 Mix the contents in Table 25 to make the correct amount of SureSelect Block mix for the number of samples used. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 45 3 Hybridization Step 1. Hybridize the library Table 25 SureSelect Block Mix Reagent Volume for 1 reaction Volume for 12 reactions (includes excess) SureSelect Indexing Block #1 (green cap) 2.5 µL 31.25 µL SureSelect Block #2 (blue cap) 2.5 µL 31.25 µL SureSelect Indexing Block #3 (brown cap) 0.6 µL 7.5 µL Total 5.6 µL 70 µL 7 In a separate PCR plate, prepare the prepped library for target enrichment. a Add 3.4 µL of 30 ng/µL prepped library to the “B” row in the PCR plate. Put each sample into a separate well. b Add 5.6 µL of the SureSelect Block Mix to each well in row B. c Mix by pipetting up and down. d Seal the wells of row “B” with caps and put the PCR plate in the thermal cycler. Do not heat the Hybridization Buffer or capture library yet, only the prepped library with blockers. e Start the thermal cycler program in Table 26. Prepped Library Figure 5 46 Prepped library shown in red SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Hybridization Step 1. Hybridize the library Table 26 3 PCR program Step Temperature Time Step 1 95°C 5 minutes Step 2 65°C Hold 8 Use a heated lid on the thermal cycler at 105°C to hold the temperature of the plate on the thermal cycler at 65°C. CAUTION The lid of the thermal cycler is hot and can cause burns. Use caution when working near the lid. 9 Maintain the plate at 65°C while you load 40 µL of hybridization buffer per well into the “A” row of the PCR plate. The number of wells filled in Row A is the number of libraries prepared. The example in Figure 6 is for 12 captures. Hybridization Buffer Prepped Library Figure 6 Hybridization buffer shown in blue Make sure that the plate is at 65°C for a minimum of 5 minutes before you go to step 10. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 47 3 Hybridization Step 1. Hybridize the library 10 Add the capture library mix from step 5 to the PCR plate: a Add the capture library mix (7 µL) to the “C” row in the PCR plate. For multiple samples, use a multi-channel pipette to load the capture library mix into the “C” row in the PCR plate. Keep the plate at 65°C during this time. b Seal the wells with strip caps. Use a capping tool to make sure the fit is tight. c Incubate the samples at 65°C for 2 minutes. 11 Maintain the plate at 65°C while you use a multi-channel pipette to take 13 µL of Hybridization Buffer from the “A” row and add it to the SureSelect capture library mix contained in row “C” of the PCR plate for each sample. (See Figure 7.) step 11 step 12 Hybridization Buffer Prepped Library SureSelect Capture Library Figure 7 SureSelect Capture Library, or “Baits”, shown in Green 12 Maintain the plate at 65°C while you use a multi-channel pipette to transfer the entire contents of each prepped library mix in row “B” to the hybridization solution in row “C”. (See Figure 7.) Mix well by slowly pipetting up and down 8 to 10 times. The hybridization mixture is now 27 to 29 µL, depending on degree of evaporation during the preincubations. 13 Seal the wells with strip caps or double adhesive film. Make sure all wells are completely sealed. Use new adhesive seals or strip caps. The structural integrity of the seals and caps can be compromised during the previous incubation steps. 48 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Hybridization Step 1. Hybridize the library CAUTION 3 Wells must be adequately sealed to minimize evaporation, or your results can be negatively impacted. If you use strip tubes, test for evaporation before you do the first experiment to make sure the tube/capping method is appropriate for the thermal cycler. Check that no more than 3 to 4 µL is lost to evaporation. 14 Incubate the hybridization mixture for 24 hours at 65°C with a heated lid at 105°C. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 49 3 Hybridization Step 2. Prepare magnetic beads Step 2. Prepare magnetic beads Use these reagents from the SureSelect Target Enrichment Kit Box #1: • SureSelect Binding Buffer • SureSelect Wash 2 1 Prewarm SureSelect Wash 2 at 65°C in a circulating water bath for use in “Step 3. Select hybrid capture with SureSelect”. 2 Vigorously resuspend the Dynabeads MyOne Streptavidin T1 on a vortex mixer. Magnetic beads settle during storage. 3 For each hybridization, add 50 µL of Dynabeads MyOne Streptavidin T1 to a 1.5-mL microfuge tube. 4 Wash the beads: a Add 200 µL of SureSelect Binding Buffer. b Mix the beads on a vortex mixer for 5 seconds. c Put the tubes into a magnetic device, such as the Dynal magnetic separator (Life Technologies). d Remove and discard the supernatant. e Repeat step a through step d for a total of 3 washes. 5 Resuspend the beads in 200 µL of SureSelect Binding Buffer. 50 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Hybridization Step 3. Select hybrid capture with SureSelect 3 Step 3. Select hybrid capture with SureSelect Use these reagents from the SureSelect Target Enrichment Kit Box #1: • SureSelect Wash 1 • SureSelect Wash 2 • SureSelect Elution Buffer • SureSelect Neutralization Buffer CAUTION Keep the Elution Buffer container tightly sealed when not in use. Prolonged exposure of SureSelect Elution Buffer to air can decrease product performance by altering the pH of the solution. 1 Estimate and record the volume of hybridization that remained after 24 hour incubation. 2 Keep the PCR plate or tubes at 65°C in the PCR machine while you add the hybridization mixture directly from the thermal cycler to the bead solution. Invert the tube to mix 3 to 5 times. Excessive evaporation, such as when less than 20 µL remains after hybridization, can indicate suboptimal capture performance. See Table 43 on page 74 for tips to minimize evaporation. 3 Incubate the hybrid-capture/bead solution on a Nutator or equivalent for 30 minutes at room temperature. Make sure the sample is properly mixing in the tube. 4 Briefly spin in a centrifuge. 5 Separate the beads and buffer on a magnetic separator and remove the supernatant. 6 Resuspend the beads in 500 µL of SureSelect Wash 1 by mixing on a vortex mixer for 5 seconds. 7 Incubate the samples for 15 minutes at room temperature. 8 Separate the beads and buffer on a magnetic separator and remove the supernatant. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 51 3 Hybridization Step 3. Select hybrid capture with SureSelect 9 Wash the beads: a Resuspend the beads in 500 µL of 65°C prewarmed SureSelect Wash 2 and mix on a vortex mixer for 5 seconds to resuspend the beads. b Incubate the samples for 10 minutes at 65°C in a recirculating water bath, heat block or equivalent. Do not use a tissue incubator. It cannot properly maintain temperature. c Invert the tube to mix. The beads may have settled. d Separate the beads and buffer on a magnetic separator and remove the supernatant. e Repeat step a through step d for a total of 3 washes. Make sure all of the wash buffer has been removed. 10 Mix the beads in 50 µL of SureSelect Elution Buffer on a vortex mixer for 5 seconds to resuspend the beads. 11 Incubate the samples for 10 minutes at room temperature. 12 Separate the beads and buffer on a magnetic separator. 13 Use a pipette to transfer the supernatant to a new 1.5-mL microfuge tube. The supernatant contains the captured DNA. The beads can now be discarded. 14 Add 50 µL of SureSelect Neutralization Buffer to the captured DNA. 52 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Hybridization Step 4. Purify the sample using Agencourt AMPure XP beads 3 Step 4. Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes. 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color. Do not freeze. 3 Add 180 µL of homogenous AMPure XP beads to a 1.5-mL LoBind tube, and add the 100 µL of cDNA library. Mix well on a vortex mixer and incubate for 5 minutes. 4 Put the tube in the magnetic stand. Wait for the solution to clear (approximately 3 to 5 minutes). 5 Keep the tube in the magnetic stand. Do not touch the beads while you carefully discard the cleared solution from the tubes. 6 Continue to keep the tube in the magnetic stand while you dispense 0.5 mL of 70% ethanol in each tube. Use fresh 70% ethanol for optimal result. 7 Let the tube sit for 1 minute to allow any disturbed beads to settle, and remove the ethanol. 8 Repeat step 6 and step 7 step once. 9 Dry the samples on the 37°C heat block for 5 minutes or until the residual ethanol completely evaporates. Do not dry the bead pellet to the point that the bead pellet appears cracked. Elution efficiency is significantly decreased when the bead pellet is excessively dried. 10 Add 30 µL nuclease-free water, mix well on a vortex mixer, and incubate for 2 minutes at room temperature. 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 12 Remove the supernatant (~30 µL) to a fresh 1.5-mL LoBind tube. You can discard the beads at this time. Stopping Point If you do not continue to the next step, store the samples at -20°C. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 53 3 54 Hybridization Step 4. Purify the sample using Agencourt AMPure XP beads SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Protocol 4 Addition of Index Tags by Post-Hybridization Amplification Step 1. Amplify the sample to add index tags 56 Step 2. Purify the sample using Agencourt AMPure XP beads 59 Step 3. Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay 60 Step 4. Assess the quantity of each index-tagged library by QPCR 62 Step 5. Pool samples for Multiplexed Sequencing 63 Step 6. Prepare sample for cluster amplification 65 This chapter describes the steps to add index tags by amplification, purify, and assess quality and quantity of the libraries, and dilute the sample appropriately for cluster amplification, and pool indexed samples for multiplexed sequencing. Agilent Technologies 55 4 Addition of Index Tags by Post-Hybridization Amplification Step 1. Amplify the sample to add index tags Step 1. Amplify the sample to add index tags Use reagents from: • Herculase II Fusion DNA Polymerase (Agilent) • SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box #2 • SureSelect RNA Primer Kit CAUTION Do not use amplification enzymes other than Herculase II Fusion DNA Polymerase. Other enzymes have not been validated. CAUTION This protocol was optimized to minimize PCR-based bias in the library preparation. To determine the number of cycles needed, do a trial amplification with 12 cycles. If you do not get enough yield for Illumina sequencing, repeat with 14 cycles. CAUTION To avoid cross-contaminating libraries, set up PCR reactions (all components except the library DNA) in a dedicated clean area or PCR hood with UV sterilization and positive air flow. Prepare 1 amplification reaction for each hybrid capture. Include a negative no-template control. To see the nucleotide sequence in each of the index included in SureSelect reagent kits, see “SureSelectXT Indexes for Illumina” on page 73. 1 For 1 library: • In a PCR tube, strip tube, or plate, prepare the reaction mix in Table 27, on ice. Mix well by gently pipetting up and down. 2 For multiple libraries: a Prepare the reaction mix in Table 27, on ice. Mix well on a vortex mixer. b Add 36 µL of the reaction mix to each well or tube. c Add 1 µL of the appropriate index PCR Primer Index 1 through Index 16 (clear caps) from the SureSelect RNA Primer Kit to each well and mix by pipetting. Use a different index primer for each sample to be sequenced in the same lane. 56 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Addition of Index Tags by Post-Hybridization Amplification Step 1. Amplify the sample to add index tags 4 d Use a pipette to add 14 µL of each DNA sample to each well or tube. Mix by pipetting. Change pipette tips between samples to avoid cross-contamination. Table 27 Herculase II Master Mix Reagent Volume for 1 reaction Captured DNA 14 µL Nuclease-free water 22.5 µL 281.25 µL 10 µL 125 µL 0.5 µL 6.25 µL Herculase II Fusion DNA Polymerase (red cap)* 1 µL 12.5 µL SureSelect ILM Indexing Post Capture Forward PCR Primer (orange cap)† 1 µL 12.5 µL PCR Primer Index 1 through Index 16 (clear caps)‡ 1 µL Total 50 µL 5X Herculase II Rxn Buffer (clear cap) * 100 mM dNTP Mix (green cap) * Volume for 12 reactions (includes excess) 437.5 µL (35 µL/reaction) * Included in the Herculase II Fusion DNA Polymerase (Agilent). Do not use the buffer or dNTP mix from any other kit. † Included in the SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box #2. ‡ Use one of the 16 primers included in the SureSelect RNA Primer Kit. 3 Put the samples in a thermal cycler with a heated lid at 105°C. Run the program listed in Table 28. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 57 4 Addition of Index Tags by Post-Hybridization Amplification Step 1. Amplify the sample to add index tags Table 28 PCR program Step Temperature Time Step 1 98°C 30 seconds Step 2 98°C 10 seconds Step 3 57°C 30 seconds Step 4 72°C 30 seconds Step 5 • Repeat Step 2 through Step 4 for a total of 12 to 16 times. Step 6 72°C 5 minutes Step 7 4°C Hold As with the pre-capture PCR amplification, minimize the number of PCR cycles used to enrich the captured DNA. The use of only half of the captured DNA for amplification lets you adjust the number of cycles by repeating the PCR if needed. Use the optimal cycle number to repeat PCR at the 50 µL reaction scale. See Table 29 for approximate number of cycles for a given library size. Results may vary based on library content. Table 29 58 Cycle times Capture Size Cycles 1 kb up to 0.5 Mb 16 cycles 0.5 Mb up to 1.49 Mb 14 cycles > 1.5 Mb 12 cycles SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Addition of Index Tags by Post-Hybridization Amplification Step 2. Purify the sample using Agencourt AMPure XP beads 4 Step 2. Purify the sample using Agencourt AMPure XP beads 1 Let the AMPure XP beads come to room temperature for at least 30 minutes. 2 Mix the reagent well so that the reagent appears homogeneous and consistent in color. Do not freeze. 3 Add 90 µL of homogenous AMPure beads to a 1.5-mL LoBind tube, and add sample library (~50 µL). Mix well on a vortex mixer and incubate for 5 minutes. 4 Put the tube in the magnetic stand. Wait for the solution to clear (approximately 3 to 5 minutes). 5 Keep the tube in the magnetic stand. Do not touch the beads while you carefully discard the cleared solution from the tubes. 6 Continue to keep the tube in the magnetic stand while you dispense 500 µL of 70% ethanol in each tube. Use fresh 70% ethanol for optimal result. 7 Let the tube sit for 1 minute to allow any disturbed beads to settle, and remove the ethanol. 8 Repeat step 6 and step 7 once. 9 Dry the samples on the 37°C heat block for 5 minutes or until the residual ethanol is completely evaporated. Do not dry the bead pellet to the point that the bead pellet appears cracked. Elution efficiency is significantly decreased when the bead pellet is excessively dried. 10 Add 30 µL nuclease-free water, mix well on a vortex mixer, and incubate for 2 minutes at room temperature. 11 Put the tube in the magnetic stand and leave for 2 to 3 minutes, until the solution is clear. 12 Remove the supernatant (~30 µL) to a fresh 1.5-mL LoBind tube. You can discard the beads at this time. Stopping Point If you do not continue to the next step, store the samples at 4°C for up to a week, or at -20°C for longer periods. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 59 4 Addition of Index Tags by Post-Hybridization Amplification Step 3. Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay Step 3. Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay NOTE As an alternative, you can use the High Sensitivity D1K ScreenTape (Agilent p/n 5067-5363) and High Sensitivity D1K Reagents (Agilent p/n 5067-5364). For more information to do this step, see the Agilent 2200 TapeStation User Manual. Use a Bioanalyzer High Sensitivity DNA Assay to assess the quality and size range. Note that the concentration of each sample loaded on the chip must be within the linear range of the assay to accurately quantify (5 pg to 500 pg). You may need to dilute your sample accordingly. Refer to the Agilent High Sensitivity DNA Kit Guide at http://www.chem.agilent.com/en-US /Search/Library/_layouts/Agilent/PublicationSummary.aspx?whid=59504. 1 Check that the 2100 Bioanalyzer electrodes have been cleaned as instructed in the reagent kit guide. 2 Open the Agilent 2100 Expert Software (version B.02.07 or higher required to run the High Sensitivity Kit), turn on the 2100 Bioanalyzer and check communication. 3 Prepare the chip, samples and ladder as instructed in the reagent kit guide. 4 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation. 5 Within the instrument context, choose the appropriate assay from the drop down list. 6 Start the run. Enter sample names and comments in the Data and Assay context. 7 Verify the results. Determine the concentration of the sample by integration under the peak. You can use the High Sensitivity Kit to quantify the amount of sample to be used for Illumina sequencing. The linear range of the High Sensitivity kit is 5 pg to 500 pg. If the reading far exceeds 500 pg, dilute and run the Bioanalyzer chip again. If the yield is too low or non-specific peaks are observed in the electropherogram, repeat the PCR with more or fewer cycles. The goal is to minimize cycles, while you produce enough library for the quantification needed for application to the flow cell. 60 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Addition of Index Tags by Post-Hybridization Amplification Step 3. Assess quality and quantity with the 2100 Bioanalyzer High Sensitivity DNA assay 4 8 Continue to sequencing. Figure 8 Analysis of Amplified Capture DNA using the High Sensitivity DNA Kit. The electropherogram shows a single peak in the size range of approximately 250 to 300 ± 20% nucleotides. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 61 4 Addition of Index Tags by Post-Hybridization Amplification Step 4. Assess the quantity of each index-tagged library by QPCR Step 4. Assess the quantity of each index-tagged library by QPCR Refer to the protocol that is included with the QPCR NGS Library Quantification Kit (Illumina GA) for more details to do this step. 1 Use the QPCR NGS Library Quantification Kit (Illumina GA) to determine the concentration of each index-tagged captured library. 2 Prepare a standard curve using the quantification standard included in the kit, according to the instructions provided in the user guide. 3 Dilute each index-tagged captured library such that it falls within the range of the standard curve. Typically this corresponds to approximately a 1:1000 to 1:10,000 dilution of the captured DNA. 4 Prepare the QPCR master mix with Illumina adaptor-specific PCR primers according to instructions provided in the kit. 5 Add an aliquot of the master mix to PCR tubes and add template. 6 On a QPCR system, such as the MX3005P, run the thermal profile outlined in the QPCR NGS Library Quantification kit user guide. Use the SYBR Green instrument setting. 7 Use the standard curve to determine the concentration of each unknown index-tagged library, in nM. The concentration will be used to accurately pool samples for multiplexed sequencing. 62 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Addition of Index Tags by Post-Hybridization Amplification Step 5. Pool samples for Multiplexed Sequencing 4 Step 5. Pool samples for Multiplexed Sequencing 1 Combine the libraries such that each index-tagged sample is present in equimolar amounts in the pool. For each library, use the formula below to determine the amount of index sample to use. V( f) × C(f) Volume of Index = --------------------------- where # × C(i) V(f) is the final desired volume of the pool, C(f) is the desired final concentration of all the DNA in the pool, for example, 10 nM for the standard Illumina protocol # is the number of index, and C(i) is the initial concentration of each index sample. Table 30 shows an example of the amount of 4 index-tagged (of different concentrations) and Low TE needed for a final volume of 20 µL at 10 nM. Table 30 Example of index volume calculation for a total volume of 20 µL Component V(f) C(i) C(f) # Volume to use (µL) Sample 1 20 µL 20 nM 10 nM 4 2.5 Sample 2 20 µL 10 nM 10 nM 4 5 Sample 3 20 µL 17 nM 10 nM 4 2.9 Sample 4 20 µL 25 nM 10 nM 4 2 Low TE 7.6 2 Adjust the final volume of the pooled library to the desired final concentration. • If the final volume of the combined index-tagged samples is less than the desired final volume, V(f), add Low TE to bring the volume to the desired level. • If the final volume of the combined index-tagged samples is greater than the final desired volume, V(f), lyophilize and reconstitute to the desired volume. 3 If you store the library before sequencing, add Tween 20 to 0.1% v/v and store at -20°C short term. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 63 4 Addition of Index Tags by Post-Hybridization Amplification Step 5. Pool samples for Multiplexed Sequencing 4 Proceed to template denaturation and flow cell preparation. Refer to the appropriate Illumina protocol. Exact library pool dilution and processing can vary based on the flow cell capacity and analysis pipeline versions being used. Refer to the appropriate Illumina user guide for instructions. This protocol has been validated with 36-base paired-end reads. However, read length can be adjusted to achieve the desired research goals. 64 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Addition of Index Tags by Post-Hybridization Amplification Step 6. Prepare sample for cluster amplification 4 Step 6. Prepare sample for cluster amplification In this step you set up cluster amplification. Conditions are optimized to provide 700K to 900K clusters/mm2 on the GAIIx and 400K to 600K clusters/mm2 on a HiSeq instrument. Genome Analyzer IIx Use reagents from the TruSeq Cluster Generation Kit appropriate for your instrument: • HT1 (Hybridization Buffer) • HP3 (2 N NaOH) 1 Dilute 30 fmol (3µL) of the 10 nM multiplexed sample pool with 16 µL of Buffer EB (10mM Tris-Cl, ph 8.5) for a total volume of 19 µL. 2 Add 1 µL of HP3 (2 N NaOH). 3 Mix the sample briefly on a vortex mixer and pulse centrifuge. 4 Incubate for 5 minutes at room temperature to denature the DNA. 5 Place the sample on ice until you are ready to proceed to final dilution. 6 Dilute 8 µL of denatured DNA with 992 µL of pre-chilled HT1 (Hybridization Buffer) for a final concentration of 12 pM. 7 Mix the sample briefly on a vortex mixer and pulse centrifuge. 8 Continue with cluster generation. Use the TruSeq SBS Kit v5–GA (36-cycle) and the appropriate Illumina multiplexed sequencing protocol. SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 65 4 Addition of Index Tags by Post-Hybridization Amplification Step 6. Prepare sample for cluster amplification HiSeq2000 with PhiX spike-in controls Use reagents from the appropriate for your instrument: • HT1 (Hybridization Buffer) • HP3 (2 N NaOH) Use the PhiX Control Kit V2 (Illumina CT-901-2001) for: • PhiX Control 1 Prepare a 1:20 dilution of HP3 (2 N NaOH) down to 0.1N NaOH. 2 Prepare 10 nM (10 fmol/µL) dilutions of the amplified capture, based on the Bioanalyzer quantitation. 3 Add 20 fmol (2 µL) of the 10 nM multiplexed sample pool into 8 µL of Buffer EB (10mM Tris-Cl, ph 8.5) to make a 2 nM solution. 4 Add 10 µL of 0.1 N NaOH. 5 Mix the sample briefly on a vortex mixer and pulse centrifuge. 6 Incubate for 5 minutes at room temperature to denature the DNA. 7 Add 980 µL of HT1 (Hybridization Buffer) to the denatured DNA to make 20 pM template solution. 8 Mix the sample briefly on a vortex mixer and pulse centrifuge. 9 Prepare 4 pM template by mixing 200 µL of 20 pM solution with 800 µL of Pre-Chilled HT1 (Hybridization Buffer). If densities higher than 400K-600K clusters/mm2 are desired, prepare a more concentrated sample from the 20 pM solution. 10 Mix the sample briefly on a vortex mixer and pulse centrifuge. 11 Remove 10 µL from solution (1000 µL) to get 990 µL. 12 Add 10 µL of PhiX Control. 13 Mix the sample briefly on a vortex mixer and pulse centrifuge. 14 Dispense 120 µL of diluted denatured sample DNA template and PhiX Control into a strip tube. 15 Place on ice until ready to use. 16 Continue with cluster generation. Use TruSeq SBS Kit–HS (50 cycle) and the appropriate Illumina multiplexed sequencing protocol. 66 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Protocol 5 Reference SureSelect Reagent Kit Content 68 Other Reagent Kits Content 70 SureSelectXT Indexes for Illumina 73 Alternative Capture Equipment Combinations 74 This chapter contains reference information. Agilent Technologies 67 5 Reference SureSelect Reagent Kit Content SureSelect Reagent Kit Content Each SureSelect Reagent Kit contains one or more of each of these individual kits: Table 31 SureSelect Reagent Kit Contents Product Storage Condition 16 96 Reactions Reactions 480 Reactions SureSelect Target Enrichment Kit Box #1 Room Temperature 5190-4393 5190-4394 5190-4395 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box #2 -20°C 5190-4455 5190-4456 5190-4457 SureSelect RNA Primer Kit -20°C 5190-5337 5500-5338 The content of each of these kits are described in the next tables. Table 32 SureSelect Target Enrichment Kit Box #1 Kit Component SureSelect Hyb #1 (orange cap, or bottle) SureSelect Hyb #2 (red cap) SureSelect Hyb #4 (black cap, or bottle) SureSelect Binding Buffer SureSelect Wash 1 SureSelect Wash 2 SureSelect Elution Buffer SureSelect Neutralization Buffer 68 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Reference SureSelect Reagent Kit Content Table 33 5 SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box #2 Kit Component SureSelect Hyb #3 (yellow cap) SureSelect Indexing Block #1 (green cap) SureSelect Block #2 (blue cap) SureSelect Indexing Block #3 (brown cap) SureSelect RNase Block (purple cap) SureSelect ILM Indexing Pre Capture PCR Reverse Primer (clear cap) SureSelect ILM Indexing Post Capture Forward PCR Primer (orange cap) Table 34 SureSelect RNA Primer Kit Kit Component SureSelect Adaptor Oligo Mix (brown cap) SureSelect Primer (brown cap) PCR Primer Index 1 through Index 16 (clear caps) SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 69 5 Reference Other Reagent Kits Content Other Reagent Kits Content These reagents are from kits other than the SureSelect Reagent kit. Make sure you use only the reagents listed here. Table 35 Herculase II Fusion DNA Polymerase (Agilent) Component DMSO (green cap) 5X Herculase II Rxn Buffer (clear cap) 100 mM dNTP Mix (green cap) Herculase II Fusion DNA Polymerase (red cap) Table 36 D1K Reagents (Agilent p/n 5067-5362) Components ladder D1K sample buffer Table 37 High Sensitivity D1K Reagents (Agilent p/n 5067-5364) Components High-Sensitivity D1K ladder High-Sensitivity D1K sample buffer Table 38 TruSeq Cluster Generation Kit* Components HT1 (Hybridization Buffer) HP3 (2 N NaOH) * Use the Illumina Cluster Generation Kit that is appropriate for your instrument and setup. See Table 5 on page 11. 70 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Reference Other Reagent Kits Content Table 39 5 PhiX Control Kit V2 (Illumina CT-901-2001) Components PhiX Control Table 40 NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S) Component NEBNext RNA Fragmentation Buffer (10X) NEBNext RNA Fragmentation Stop Solution (10X) Linear Acrylamide (10 mg/mL) Random Primers (3 µg/µL) Murine RNase Inhibitor NEBNext First Strand Synthesis Reaction Buffer (5X) NEBNext Second Strand Synthesis Enzyme Mix NEBNext Second Strand Synthesis Reaction Buffer (10X) Phosphorylation Reaction Buffer (10X) Deoxynucleotide Solution Mix (10 mM each dNTP) T4 DNA Polymerase DNA Polymerase I, Large (Klenow) Fragment T4 Polynucleotide Kinase Deoxyadenosine 5’- Triphosphate (dATP) (1.0 mM) Klenow Fragment (3’ → 5’ exo-) NEBuffer 2 for Klenow Fragment (3’ → 5’ exo-) (10X) Quick T4 DNA Ligase Quick Ligation Reaction Buffer (2X) Nuclease-free water SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 71 5 Reference Other Reagent Kits Content Table 40 NEBNext mRNA Sample Prep Reagent Set 1 (New England Bioscience p/n E6100S) (continued) Component PhusionR High-Fidelity DNA Polymerase (manufactured by Finnzymes Oy) PhusionR HF Buffer (5X) (manufactured by Finnzymes Oy) Table 41 QIAquick PCR Purification Kit (Qiagen p/n 28104) Components QIAquick spin column Buffer PB* Buffer PE (concentrate) Buffer EB pH Indicator I collection tube (2 mL) loading dye * Contain chaotropic salts which are irritants. Take appropriate laboratory safety measures and wear gloves when handling. 72 SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing Reference SureSelectXT Indexes for Illumina 5 SureSelectXT Indexes for Illumina The nucleotide sequence of each of the SureSelectXT index is listed in Table 42. Table 42 SureSelectXT Indexes 1-16 Index Number Sequence 1 ATCACG 2 CGATGT 3 TTAGGC 4 TGACCA 5 ACAGTG 6 GCCAAT 7 CAGATC 8 ACTTGA 9 GATCAG 10 TAGCTT 11 GGCTAC 12 CTTGTA 13 AAACAT 14 CAAAAG 15 GAAACC 16 AAAGCA SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing 73 5 Reference Alternative Capture Equipment Combinations Alternative Capture Equipment Combinations Table 43 lists combinations of thermal cycler, lid temperature, plates or strips, and sealing method (strip caps or sealing tape) other than those used in this protocol that have shown minimal evaporation. Refer to this list for additional of equipment combination options for hybridization. Note that minimal evaporation is needed to ensure good capture results. Table 43 Tested options that show minimal evaporation PCR Machine Plate/Strips Cover Comments Agilent Mx3005P QPCR Mx3000P Strip Tubes (401428) MX3000P Optical Strip Caps (401425) Heated Lid Agilent Mx3005P QPCR MicroAmp Optical 96-well reaction plate (N801-0560) MicroAmp Clear Adhesive Film (4306311) Heated Lid; ABI compression pad (4312639) Use two layers of film. ABI GeneAmp 9700 MicroAmp Optical 96-well Reaction Plate (N801-0560) MicroAmp Caps (8caps/strip) (N801-0535) Heated Lid ABI Veriti (4375786) MicroAmp Optical 96-well Reaction Plate (N801-0560) MicroAmp Clear Adhesive Film (4306311) Heated Lid; ABI compression pad (4312639) Use two layers of film. 74 Eppendorf Mastercycler Eppendorf 8-Tube PCR Tubes Attached lids Lid heating set to 75°C BioRad (MJ Research) PTC-200 Agilent strip tubes 410022 (Mx4000) Agilent Optical cap 410024 (Mx4000) Heated Lid BioRad (MJ Research) PTC-200 Agilent strip tubes 410022 (Mx4000) Agilent Optical cap 401425 (Mx3000/3005) Heated Lid BioRad (MJ Research) PTC-200 Agilent 96-well Plate 410088 (Mx3000/3005) Agilent Optical cap 401425 (Mx3000/3005) Heated Lid SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing www.agilent.com In This Book This guide contains information to run the SureSelect RNA Target Enrichment for Illumina Paired-End Multiplexed Sequencing protocol. © Agilent Technologies, Inc. 2011-2012 Version 2.2.1, February 2012 *G7580-90010* G7580-90010 Revision B2 Agilent Technologies
